Please refer to the corresponding frequently asked questions (FAQs) below.
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Do you have a specific protocol used for the application of PepSpot™ membranes?
JPT Peptide Technologies provides a detailed protocol together with every PepSpot™ peptide membrane.
How does JPT Peptide Technologies QC the synthesis of membrane-bound peptides?
In addition to the membrane bound PepSpot™ peptides ordered, JPT Peptide Technologies assembles control peptides on the same membrane, which are connected via a cleavable linker system to the cellulose surface.
After cleavage of these peptides from the membrane, their quality will be checked by MALDI-MS. The control peptides include specific sequences which are difficult to synthesize.
The purity of the peptides synthesized varies for each peptide and is dependent on the sequence and length.
Our analyses have determined that peptide purity is typically >70% for average 6-15 mers (by HPLC and mass spectroscopy).
Which end of the peptides is coupled to the cellulose membrane?
The peptides are covalently linked to the cellulose support via the C-terminus. For sufficient flexibility for binding studies, a two ß-alanine residue spacer or a short PEG spacer is inserted between the cellulose and each PepSpot™ peptide.
When do you recommend acetylation of the N-terminus?
N-terminal acetylation is recommended for peptide scans, because peptides are more stable to degradation and the uncharged N-acetyl better represents the region in the native antigen than a charged NH3+-group. There is no extra charge for acetylation.
Do you recommend to substitute cystein residues by serine residues?
Yes, as cysteine containing peptides are sensitive to oxidation and cyclization. Therefore, the substitution of Cys by Ser increases the peptide quality. Secondly, unspecific interactions between cysteine residues and antibodies/proteins could occur which give false-positive results. Finally, Cysteine is very scarcely an essential residue in antibody epitopes.
How are the data from the PepSpot™ incubation evaluated?
Detections can be performed with a chemiluminescence substrate in combination with an imaging system or, if not available, with a standard X-ray film and film cassette. Detections via fluorescence read-out are not recommended.
Which enzyme-labeled antibodies are recommended for detection ?
We recommend the use of horseradish peroxidase(HPR)-conjugated antibodies, while other enzymes such as alkaline phosphatase (AP) may also be used.
Please note: It has often been observed that AP binds to peptide spots depending on their sequences, whereas HRP in almost all cases did not interact with the peptides.
Please also note: Do not use sodium azide as a preservative for buffers with peroxidase as it is an inhibitor of the enzyme.
Can unnatural or modified amino acids be used in the synthesis?
The spot synthesis technology is compatible with D and L forms of amino acids as well as unnatural amino acids.
However, the chemistry requires that the amino acids have an Fmoc protection group at their N-terminus and acid labile protecting groups on their side chains (e.g. Pbf, OtBu, Trt, Boc, tBu, etc).
Please note: Some unnatural amino acids may give poor coupling yields due to sterical hindrance.
Can the attached peptides be cyclized or biotinylated?
Yes, JPT Peptide Technologies also offers Cys-Cys-cyclization and biotinylation for PepSpots™.
What length of offset do I need for peptide scans?
Most linear peptide epitopes for antibody applications are between 3-9 amino acids in length.
To be on the save side, we recommend a peptide length of 13 amino acids and an offset of 2 to 3 amino acids for the mapping of linear epitopes.
Hence, when working with a large protein, it is recommended that the offset does not exceed 3 amino acids in order to localize the epitope.
An offset of 1 is recommended for finer mapping of the epitope. For the mapping of conformation dependent (discontinuous) epitopes, we recommend a peptide length of 15 to 20 amino acids and an offset 5 amino acids in maximum.
How much peptide is synthesized per Spot?
The amount of peptide that is synthesized is determined theoretically from calculating the amount of free amines available for coupling per Spot and assuming a coupling efficiency of >98% per cycle.
A typical synthesis is expected to yield between 5-10 nmol (6-12 µg for an average 10mer peptide).
What is the size of a PepSpots™ peptide?
The PepSpots™ peptides have a diameter of approx. 2-3 mm.
What is the density of peptides on the membrane?
The peptides are synthesized in a grid fashion with 0.37 cm x 0.37 cm (0.15 in x 0.15 in, center-to-center) spacing, 20 peptides per row. Other formats are available upon request.
What is the chemical stability of the attached peptides?
JPT Peptide Technologies offers two types of PepSpots™ peptide membranes:
- PepSpots™: The connection between the peptide and the membrane is an ester bond and therefore labile towards strong bases (i.e. NaOH) and nucleophiles (i.e. NH2R).
The peptides themselves are quite stable.
However, strong acids may occasionally break acid labile amide bonds such as Asp-Pro. In addition, there are several amino acids such as Cys or Met which are sensitive towards oxidation.
Finally, very strong bases (NaOH) may hydrolize peptide bonds as well.
- PepSpots™ (PEG): The connection between the peptide and the membrane is an ether bond and therefore stabile towards strong bases (i.e. NaOH) and nucleophiles (i.e. NH2R).
We suggest to use this membrane type if electroblotting experiments or application of alkaline phosphatase is planned.
What is the chemical stability of the membranes?
The membrane material is stable to synthesis conditions, but should not be exposed to strong acids for prolonged periods.
What are the appropriate storage conditions for the membrane?
New membranes should be stored at -20°C until use.
Incubated membranes which will be reused within several days should be kept with a small volume of T-TBS buffer in a Petri dish at 4°C.
Incubated membranes which will be stored for a longer period should be regenerated and kept at -20°C.